The use of genetic information in the treatment of disease is one the most promising avenues of medical research. There are different approaches of delivering genetic information to a patient; most prominently among them viral vectors of different origins and “naked” DNA vectors propagated in bacteria. The use of viral vectors has been discredited of late because of safety considerations.
The use of bacterially derived plasmids in the clinic increases the importance of effective and economical means and methods of manufacturing and purifying large amounts of plasmid DNA to very high standards of purity. In order to satisfy the criteria of pharmaceutical manufacturing adopted by most authorities, a method for the purification also needs to yield reproducible and validated results.
A number of methods have been reported for the purification of pharmaceutical grade plasmid DNA. Most of them follow a similar scheme, involving a first lysis step, in which the bacteria are broken down, a subsequent denaturation step that destroys nucleic-acid interactions with proteins, and finally a procedure by which the target nucleic acid content is derived in a sequence of precipitation steps and at least one chromatographic step.
The quality obtained by these purification methods is variable. One feature, however, is that certain substances present in the bacterial biomass, among them polysaccharides derived from the bacterial cell wall, lipopolysaccharides and RNA, are difficult to remove without several chromatographic steps, and tend to contaminate the standard DNA preparations. Some of these bacterially derived contaminants are extremely potent effectors of various defence systems of higher eukaryotes, possibly because of their intrinsic function as a signal of bacterial infection. The elimination of these contaminants is a major problem in the manufacture and purification of plasmid DNA.
Ultrafiltration of biological products is known. A method for obtaining a protein from tissue fluid by diafiltration is shown in WO 98/54195. Many methods for the isolation and purification of nucleic acids are also known and taught in different patents and patent applications. For example, WO98/05673 describes a process for producing highly purified DNA which involves a combination of diafiltration and chromatography steps. Diafiltration is used before and after ion exchange chromatography primarily to ensure that the nucleic acid containing sample is in the correct buffer. A key feature of this proposal is that the diafiltration uses an open-channel or hollow fibre ultrafiltration unit to prevent turbulence at the ultrafiltration membrane so as to obtain a gel layer.
AU 723817 (derived from WO98/30685) also concerns a method for producing a highly purified plasmid DNA. In this method, two column chromatography steps are used; a Q-sepharose® chromatography step followed by a hydroxylapatite chromatography step. The eluate from the hydroxylapatite chromatography step is subjected to cross-flow filtration which is stated in the disclosure to be essential. This step is used to remove salt and other contaminants from the column eluate.
WO 00/05358 is primarily directed to the use of a static mixer to obtain a lysed cell solution in a method for purifying plasmid DNA. This, too, relies upon an ion exchange chromatography step to purify plasmid DNA and diafiltration by tangential flow using an open channel ultrafiltration unit is employed to ensure that the plasmid DNA is in the correct buffer.
U.S. Pat. No. 4,623,723 is directed to the separation of crude nucleic acid solutions using hollow fibre ultrafiltration membranes. It is proposed to separate RNA from DNA using diafiltration against deionised water. No data are given as to the effectiveness of the method.
In order to achieve highly purified pharmaceutical grade plasmid DNA, each of the above methods is primarily reliant upon a chromatographic step to remove contaminants. This can increase the time for purification, especially where multiple chromatographic steps are required, and will increase the cost involved as well.